We exploited the high sensitivity of fluorescence measurements to determine whether alpha-synuclein (alpha-syn) and glucocerebrosidase (GCase) can interact. This approach was critical for the success of this project because GCase supply is limited. We then were able to carry out nuclear magnetic resonance (NMR) studies. Our NHGRI colleagues used immunoprecipitation (IP) and immunofluorescence studies to study this interaction in patient brain samples and neuronal cell culture, respectively. Lastly, we examined the effect of N370S mutation on complex formation. N370S is a common GD mutation, a catalytically compromised enzyme found in up to 4% of the Ashkenazi Jewish population and has clearly been associated with an increased PD risk. For the first time, we showed that GCase interacts with alpha-syn; the complex does not form at pH 7.4 and is stabilized by electrostatics at pH 5.5, with dissociation constants (Kd) ranging from 2-22 microM in the presence of 25-100 mM NaCl. While a micromolar Kd value is considered modest for well-folded proteins, it is quite reasonable for an intrinsically disordered protein (IDP). One of the unique features of an IDP is the ability to have specific interactions yet moderate binding affinity. Moreover, the neuronal concentration of alpha-syn is estimated to be quite high up to tens of microM; thus, a biologically relevant interaction at this strength is plausible. We also confirmed the association from the perspective of GCase, using Forster energy transfer from intrinsic Trp residues of GCase to dansyl-labeled alpha-syn. The affinity for N370S mutant is reduced by two-fold compared to the wild-type enzyme. Since the Ser mutation only alters GCase structure near its active site, we hypothesize that the alpha-syn C-terminus is binding near the active site. Of note, the structure for GCase bound to the inhibitor, conduritol-beta-epoxide (CBE), also adopts a similar conformation as the N370S mutant. A weaker protein-protein interaction should be observed in the presence of CBE if our assertion is correct. Indeed, CBE-bound GCase exhibits comparable affinity as N370S. In both structures, loop 1 adopts an extended structure with residue D315 partially surface-exposed, whereas it is buried in the WT structure. Thus, the weaker binding could be rationalized by electrostatic repulsion between D315 and the acidic alpha-syn C-terminal tail. To map this intermolecular interaction in detail, we isotopically labeled alpha-syn (15N) for NMR spectroscopy which provides a residue-by-residue characterization by monitoring all backbone amide hydrogen and nitrogen resonances for each non-proline residue. Specifically, we identified alpha-syn residues 118-137 as the binding site. This region contains eight acidic residues as well as three tyrosines, allowing for both electrostatic and hydrophobic interactions. Correspondingly, there are numerous positively charged side chains on the GCase surface that can provide complementary electrostatic contacts upon binding. In vivo interactions between the endogenous proteins were verified by IP of GCase from brain autopsy samples. GCase-positive bands were identified by Western blot analysis in samples from the PD patient(GBA genotype: WT/WT), carrier with PD(N370S/WT), and PD patient with type 1 GD(N370S/N370S). As expected, the GCase-positive band was not present in the type 2 patient with negligible GCase. In accord with the recombinant proteins, the interaction between human brain tissue derived alpha-syn and GCase is observed only at the acidic pH, but is lost, or not measurable, at neutral pH. To investigate the intracellular localization of GCase and alpha-syn, a dopaminergic human neuroblastoma BE(2)-M17 cell model was established where both proteins are stably over-expressed. Co-localization of GCase and alpha-syn was observed in distinct cellular inclusions, which also stained positive for cathepsin D, a mannose-6-phosphate dependent specific marker of acidic lysosomal compartments, suggesting their close proximity in the lysosome. While our data do not preclude protein-protein interactions in other cellular milieux, we suggest that the lysosome is a primary site of interaction for alpha-syn and GCase. Because the lysosome plays an important role in protein degradation, this newly identified interaction between alpha-syn and GCase can potentially influence alpha-syn homeostasis in neurons. Taken together with the genetic connection between GD and PD, the results imply that an altered alpha-syn-GCase interaction in the lysosome could perturb this equilibrium and set the stage for PD progression. We hypothesize that this interaction to be beneficial by promoting lysosomal degradation of alpha-syn, or inhibiting adverse alpha-syn aggregation. Thus, mutations that decrease the amount of enzyme reaching the lysosome or weaken the interaction, as seen with the N370S mutant, could reduce this benefit and increase the probability of dysfunction. We believe that this alpha-syn-GCase interaction provides the groundwork to expand our studies in exploring molecular mechanisms linking PD with mutant GBA alleles.